Role of a bacterial glycolipid in Sec-independent membrane protein insertion

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.

coupling protocol using Fmoc-amino acid/N, N-diisopropylcarbodiimide (DIC)/ethyl 2cyano-2-(hydroxyimino)acetate (OxymaPure ® ) 1 . Boc-Thr(Fmoc-Val)-OH, an O-acyl isodipeptide unit, was introduced into the Val 8 -Thr 9 bond 2,3 . After construction of the protected peptide resin, the O-acyl isopeptide was cleaved by a trifluoroacetic acid (TFA) cocktail, purified by high-performance layer chromatography (HPLC), and applied to the NMR study with in situ O-to-N acyl migration (Fig. S1) to produce the native target peptide 4 . The mass spectrum (MS) of the O-acyl isopeptide was observed using an Agilent G1956B LC/MSD detector using an Agilent 1100 series HPLC system, and the observed mass (most abundant masses) was derived from the experimental m/z value for the protonation state of the target peptide. Electrospray ionization MS: [M+H] + calculated for C213H351N48 15 N5O59S was 4634.6, while the measured value was found to be 4634.7.
Pf3_24_1 for the 15

Preparation of the membrane sample
To prepare the membrane sample, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid For the membrane insertion experiments (Figs. 3, 4, S7, S10, and S11), we first prepared large unilamellar vesicles (LUVs). Phospholipids (34.5 mol) were solubilized in chloroform in the absence and presence of MPIase (mini-MPIase-3 [5 mol% in total lipid amount] or natural MPIase [1 mol% in total lipid amount]) and/or diacylglycerol (DAG; 5 mol% in total lipid amount). After complete solvent evaporation, the resulting lipid film was hydrated with 400 L of HEPES buffer and vortexed. The suspension was freeze-thawed for ten cycles and extruded through 100-nm polycarbonate filters (Avestin, Ottawa, ON, Canada). Then, Pf3_24_3 (0.77 mol) solubilized with 200 L of DHPC (6.9 mol) solution was mixed with LUV and incubated for 30 min at 37 °C. This mixture was diluted 20 times with HEPES buffer until the DHPC concentration reached below the critical micelle concentration (CMC) of DHPC (1.6 mM) 5,6 . The supernatant containing DHPC was removed after centrifugation, and this step was repeated twice to remove DHPC. For all NMR measurements, samples were packed within a 4-mm NMR tube that was closed tightly with a seal cap to prevent drying (Phi Creative, Kyoto, Japan).
To measure fluorescence (Fig. 4, S8, and S9), phospholipids (0.1 mol for packing analysis or 2.72 mol for anisotropy measurement) were solubilized in chloroform in the absence and presence of natural MPIase (1 mol% in total lipid amount) and/or DAG (5 mol% in total lipid amount). 6-Lauroyl-2-dimethylamino naphthalene (Laurdan; 1 nmol for packing analysis) or 1,6-diphenyl-1,3,5-hexatriene (DPH; 2 nmol for anisotropy measurement) from an ethanol stock was added before drying the samples. After complete evaporation of the solvent, the lipid film was hydrated with 1 mL of HEPES buffer (final lipid concentration of 0.1 mM) and vortexed extensively. The suspension was freezethawed for ten cycles and transformed into 100 nm LUVs using an extruder (Avestin).

Measurement of solid-state NMR
All solid-state NMR measurements were carried out using a Bruker Avance III 600 (Bruker Biospin, AG, Switzerland) equipped with a narrow-bore magnet operated at a 1 H resonance frequency of 600 MHz. Data were recorded using a 4-mm E-free triple-  (Figs. 3, S7, S10, and S11) were acquired at -5 C under 5-kHz MAS. The signal-to-noise ratio (SNR) of spectral peaks (Figs. 3, S7, S10, and S11) were calculated by the program "sinocal" in Topspin 3.1 (Bruker Biospin, AG, Switzerland). Subsequently, the errors of the membrane insertion efficiency x in Eq. 1 due to the spectral noise were estimated by calculating the difference in x between two cases in which relative peak intensities at 125 ppm (I 125 ) and 120 ppm (I  ) fluctuate due to the noise from I  (1+0.5/SNR) and I  (1-0.5/SNR)) to I  (1-0.5/SNR) and I  (1+0.5/SNR). achieved using the FSLG sequence with a transverse field of 71.4 kHz. The 1 H chemicalshift scaling factor due to the FSLG sequence was calibrated on the glycine molecule to 0.72. All the temperatures quoted are the calibrated temperatures. The 15 N chemical shifts were externally referenced to the methionine amide resonance of N-formyl-Met-Leu-Phe-OH (127.9 ppm) 9 .

Data analysis of SAMPI4 spectra
SAMPI4 spectra were calculated using scripts available on a website

Steady-state fluorescence measurement
Fluorescence measurements were performed using a Duetta fluorescence spectrophotometer (Horiba Scientific, Kyoto, Japan). The fluorescence emission spectra In the DPH anisotropy measurements (Fig. S9) where IVV and IVH are the emission intensities measured in the parallel and perpendicular directions to the exciting beam, respectively, and G is the grating factor (G = IVH/IHH).
The addition of 0.1 mol% DPH to EPL LUV increased membrane insertion by 3%. Thus, actual fluorescence anisotropy values on the horizontal axis in the correlation plot ( Fig.   4b) are expected to shift almost uniformly and infinitesimally to the smaller value in the absence of DPH, but the slope of the global linear fits would hardly be affected.

CD spectroscopy
CD spectra of Pf3_24_3 (Fig. S12) were recorded at 37 C on a Jasco J-725 spectropolarimeter (Jasco, Tokyo, Japan) using a 1-mm-path-length cell. The spectra were measured between 190 and 250 nm, and the average blank spectra were subtracted. Data were collected at 0.1 nm with a scan rate of 100 nm/min and a time constant of 0.5 s. The peptide concentration was 383 M. Eight scans were averaged for each sample, and the 8 appropriate background contribution was determined.

Molecular dynamics simulation
We explored MPIase conformation (Fig. S3)            LUV, which was co-solubilized with DMPC before making the LUV membranes (dark red). Solubilized with a DHPC solution (green); Pf3_24_3 in HEPES buffer (blue), which was prepared without using LUVs, as shown in Fig. 2a. These spectra were obtained after subtraction of the background CD spectra. All measurements were recorded at 37 °C.